ABSTRACT Our proposed studies will investigate the central hypothesis that epigenetic enzymes regulate lineage- commitment and maturation of bone forming cells in vivo. Our studies will inform new strategies for bone anabolic therapies aimed at reducing fracture risk. Current therapies have major clinical side-effects and patient restrictions. Yet, with the aging demographics of the US population, there is a significant urgency to develop new strategies that promote bone accrual. In addition, there are a range of short term anabolic applications (e.g., spine fusion, radiation induced bone loss) that would benefit from new mechanism-based approaches. The proposed work will provide (i) proof of concept for leveraging epigenetic drugs to promote bone accrual in a pre-translational setting, but also (ii) fundamental insights into the role of chromatin heterochromatinization in controlling bone formation. Aim 1 will assess the in vivo bone stimulatory effects of conditional ablation of the gene for histone methyl transferase EZH2 during post-natal development and in skeletally mature mice in which the EZH2 gene is deleted in mesenchymal stem cells or osteoblasts. These studies will reveal whether genetic loss of EZH2 stimulates bone accrual, is osteoprotective (after estrogen-depletion in ovariectomized mice), and/or accelerates bone healing. These genetic studies will follow-up on our preliminary data indicating that pharmacological inhibitors with established safety profiles (e.g., EZH2 inhibitor GSK126) can be used to promote bone accrual in vivo and osteoblast differentiation in vitro. Aim 2 will examine the molecular basis for the bone stimulatory effects of EZH2 inactivation. Using genomics approaches (ChIP-seq and RNA-seq), we will define and functionally test signaling molecules and gene regulatory factors that respond to EZH2 inhibition. We will also specifically investigate the attractive model that EZH2 inhibition initiates a sustained bone anabolic response by enhancing endogenous paracrine signaling of osteogenic ligands (e.g., BMP, WNT and PTH). We will focus specifically on examining the molecular basis for our new observation that GSK126 and BMP2 synergize in osteoblast maturation. Our findings have important clinical ramifications by generating genetic support for considering epigenetic drugs in promoting new bone formation. Conceptual innovation of these studies will be obtained by molecular analysis of the epigenetic regulatory role for EZH2 which permits definition of early mechanistic events that control osteogenic differentiation and osteoblast maturation.